Cytosolic pool

Several additional points should be made. First, although oxygen esters usually have lower group-transfer potentials than thiol esters, the O—acyl bonds in acylcarnitines have high group-transfer potentials, and the transesterification reactions mediated by the acyl transferases have equilibrium constants close to 1. Second, note that eukaryotic cells maintain separate pools of CoA in the mitochondria and in the cytosol. The cytosolic pool is utilized principally in fatty acid biosynthesis (Chapter 25), and the mitochondrial pool is important in the oxidation of fatty acids and pyruvate, as well as some amino acids. 

Several groups of drugs that bind to tubulin at different sites interfere with its polymerization into microtubules. These drugs are of experimental and clinical importance (Bershadsky and Vasiliev, 1988). For example, colchicine, an alkaloid derived from the meadow saffron plant Colchicum autumnale or Colchicum speciosum), is the oldest and most widely studied of these drugs. It forms a molecular complex with tubulin in the cytosol pool and prevents its polymerization into microtubules. Other substances such as colcemid, podophyllotoxin, and noco-dazole bind to the tubulin molecule at the same site as colchicine and produce a similar effect, albeit with some kinetic differences. Mature ciliary microtubules are resistant to colchicine, whereas those of the mitotic spindle are very sensitive. Colchicine and colcemid block cell division in metaphase and are widely used in cytogenetic studies of cultured cells to enhance the yield of metaphase plate chromosomes. 

Another drug is taxol, which is extracted from the bark of the Pacific yew tree, Taxus brevijolia. Unlike colchicine and the vinca alkaloids, taxol binds tightly to microtubules and stabilizes them against depolymerization by Ca. It also enhances the rate and yield of microtubule assembly, thereby decreasing the amount of soluble tubulin in the cytosol pool. Again, the overall effect of taxol is to arrest dividing cells in mitosis. Taxol is used in cancer chemotherapy. 

The diversity of these subcellular actin structures is remarkable and appears to be determined by the interactions of many actin-binding proteins (ABPs) as well as by changes in the concentrations of intracellular signaling molecules such as Ca and cAMP, by small GTP-binding proteins, and by signals arising from mechanical stress. Approximately 50% of the actin molecules in most animal cells are unpolymerized subunits in the cytosolic pool and exist in a state of dynamic equilibrium with labile F-actin filamentous structures (i.e., new structures are formed while existing structures are renewed) (Hall, 1994). 

This three-step process for transferring fatty acids into the mitochondrion—esterification to CoA, transesterification to carnitine followed by transport, and transesterification back to CoA—links two separate pools of coenzyme A and of fatty acyl-CoA, one in the cytosol, the other in mitochondria These pools have different functions. Coenzyme A in the mitochondrial matrix is largely used in oxidative degradation of pyruvate, fatty acids, and some amino acids, whereas cytosolic coenzyme A is used in the biosynthesis of fatty acids (see Fig. 21-10). Fatty acyl-CoA in the cytosolic pool can be used for membrane lipid synthesis or can be moved into the mitochondrial matrix for oxidation and ATP production. Conversion to the carnitine ester commits the fatty acyl moiety to the oxidative fate. 

As we noted in Chapter 16, the enzymes of many metabolic pathways are clustered (p. 605), with the product of one enzyme reaction being channeled directly to the next enzyme in the pathway. In the urea cycle, the mitochondrial and cytosolic enzymes appear to be clustered in this way. The citrulline transported out of the mitochondrion is not diluted into the general pool of metabolites in the cytosol but is passed directly to the active site of argininosuccinate synthetase. This channeling between enzymes continues for argininosuccinate, arginine, and ornithine. Only urea is released into the general cytosolic pool of metabolites. 

FIGURE 19-1 Biochemical anatomy of a mitochondrion. The convolutions (cristae) of the inner membrane provide a very large surface area. The inner membrane of a single liver mitochondrion may have more than 10,000 sets of electron-transfer systems (respiratory chains) and ATP synthase molecules, distributed over the membrane surface. Heart mitochondria, which have more profuse cristae and thus a much larger area of inner membrane, contain more than three times as many sets of electron-transfer systems as liver mitochondria. The mitochondrial pool of coenzymes and intermediates is functionally separate from the cytosolic pool. The mitochondria of invertebrates, plants, and microbial eukaryotes are similar to those shown here, but with much variation in size, shape, and degree of convolution of the inner membrane. 

Answer The transport of fatty acid molecules into mitochondria requires a shuttle system involving a fatty acyl-carnitine intermediate. Fatty acids are first converted to fatty acyl-CoA molecules in the cytosol (by the action of acyl-CoA synthetases) then, at the outer mitochondrial membrane, the fatty acyl group is transferred to carnitine (by the action of carnitine acyl-transferase I). After transport of fatty acyl-carnitine through the inner membrane, the fatty acyl group is transferred to mitochondrial CoA. The cytosolic and mitochondrial pools of CoA are thus kept separate, and no labeled CoA from the cytosolic pool enters the mitochondrion. 

Answer Pyruvate dehydrogenase is located in the mitochondrion, and glyceraldehyde 3-phosphate dehydrogenase in the cytosol. Because the mitochondrial and cytosolic pools of NAD are separated by the inner mitochondrial membrane, the enzymes do not compete for the same NAD pool. However, reducing equivalents are transferred from one nicotinamide coenzyme pool to the other via shuttle mechanisms (see Problem 21). 

Upon stimulation by an agonist but not an antagonist, PKC redistributes from the cytosolic pool to the particulate fraction. This redistribution could be measured in vivo [102] and in vitro [103-105] and was shown to be dependent upon Ca2+ influx [102]. These data suggest that a minimal intracellular concentration of Ca2+ is required for adequate PKC stimulation by DAGs. Since DAG production and translocation of PKC are parallel events it appears that PKC mediates endogenous DAG stimulation of LH secretion. 

Compartmentation of glutathione has been demonstrated in that a separate pool of glutathione exist in the cytoplasm from that in the mitochondria (Figure 18.6). The cytosolic pool of glutathione has been characterized in terms of cellular protection (Table 18.1). 

Dissociation of GDP may be inhibited by specific proteins known as guanine nucleotide dissociation inhibitors (GDI). Proteins with this function are found in members of the superfamily of Ras proteins (see Chapter 9). The GDIs have the function, above all, to provide a cytosolic pool of inactive, GDP-bound proteins (see Section 9.1). 

Rab proteins exist in all cells and form the largest branch of the Ras superfamily. This family performs a central function in vesicular transport. Rab proteins influence and regulate the budding, targeting, docking and fusion of vesicles as well as processes of exocytosis and endocytosis involving clathrin-coated vesicles. During these functions, Rab proteins cycle between the cytosol and the cell membrane, and this cyle is superimposed on a GDP/GTP cycle. The cytosolic pool of the Rab pro-... 

In addition to synthesis of new transmitter, NE stores are also replenished by transport ofNE previously released to the extracellular fluid by the combined actions of a NE transporter (NET, or uptake 1) that terminates the synaptic actions of released NE and returns NE to the neuronal cytosol, and VMAT-2, the vesicular monoamine transporter, that refills the storage vesicles from the cytosolic pool ofNE ("see below). In the removal ofNE from the synaptic cleft, uptake by the NET is more important than extraneuronal uptake (ENT, uptake 2). The sympathetic nerves as a whole remove -87% of released NE via NET compared with 5% by extraneuronal ENT and 8% via diffusion to the circulation. By contrast, clearance of circulating catecholamines is primarily by nonneuronal mechanisms, with liver and kidney accounting for >60% of the clearance. Because VMAT-2 has a much higher affinity for NE than does the metabolic enzyme, monoamine oxidase, over 70% of recaptured NE is sequestered into storage vesicles. 

According to Halestrap [98] activation of mitochondrial electron transport not only increases the proton-motive force but also the intramitochondrial ATP concentration which is important for intramitochondrial ATP utilising reactions like pyruvate carboxylation and citrulline synthesis, processes known to be activated by glucagon. Siess et al. [35], however, showed that in hepatocytes glucagon not only increased mitochondrial ATP, but also the sum of ATP, ADP and AMP at the expense of the cytosolic pool of adenine nucleotides, a phenomenon to which no attention has been paid in the literature. Exchange between ADP and ATP cannot increase the mitochondrial adenine nucleotide pool. Possibly net influx of adenine nucleotides can occur via exchange between ADP and mitochondrial phosphoenol-pyruvate (see [4] for literature). 

The overt carnitine palmitoyltransferase activities in each fraction were measured with an optimal concentration of palmitoyl-CoA (above). A previous report suggested that palmitoyl-CoA is not a good substrate for overt CPT in microsomes. However, we have not been able to confirm this, as the palmitoyl-CoA requirement for the enzymes in all three membranous fractions was very similar to that described previously for mitochondrial outer membrane CPT Peroxisomes displayed the highest specific activity of overt CPT (see below) and their overall contribution, computed on a per gram liver basis was appreciable and of the same order of that in microsomes, which had a relatively low specifie aetivity, but which occurred at much higher protein densities within the cell. Mitochondria accoimted for about 65% of total CPT activity. These estimates are very similar to those reported by. It is evident, therefore, that the peroxisomal and microsomal forms of overt CPT constitute an important component of the overall CPT activity with access to the cytosolic pools of acyl-CoA and malonyl-CoA. 

Acetyl-CoA has already been mentioned as a key precursor for many industrially relevant compounds. For example, it is a direct precursor for the mevalonate pathway to obtain isoprenoids. It is also a key precursor for malonyl-CoA, yielding the production of fatty acids (biodiesel) and polyketides [19]. The challenge of engineering the acetyl-CoA availability in yeast lies in its compartmentalization. While acetyl-CoA is readily available in the mitochondrium, the cytosolic pool is low. The cytosolic pool of acetyl-CoA is fed from acetate, which is activated by a bond to coenzyme A at the expense of 1 ATP. It becomes therefore obvious that any metabolic pathway using cytosolic acetyl-CoA aiming at mass production is energetically detrimental and inefficient - if not recombinantly redesigned [20]. 

Sea urchin male pronuclei formed in vitro under the conditions described in this chapter remain small ( 4 /tm in diameter) and do not contain a lamina. Pronuclear swelling is promoted only if extra ATP is added to the egg extract. Nuclear swelling has been shown to be associated with, and dependent on, assembly of a nuclear lamina from a cytosolic pool of soluble lamins (Collas et al., 1995). More recent unpublished observations have shown that B-type lamins are also associated with a minor fraction of MV2i3 vesicles. The contribution of these vesicle-associated lamins to the nuclear lamina is under investigation. In the surf clam, sperm chromatin decondensation and pronuclear expansion are continuous processes that occur in parallel with nuclear envelope and lamina assembly in 65-min-activated egg extracts (Longo et al., 1994). Manipulations of this in vitro system to control each of these processes have not been reported. 

A small pool rapidly equilibrating with exogenous amino acids directly provides precursors for protein synthesis (cytosolic pool, internal pool, cf, ref, 6),... 

In fungal cells (Neurospora, yeasts) the expandable pool was shown to be localized in vacuoles which can be isolated (4, 10) and contain their own specific transport system in the tonoplast membranes (10 Thus vacuoles are main storage sites of amino acids of fungal (and plant ) cells. Further, they may function as dominant regulatory agents, if uptake and release are responsive to changes in cytosolic pool sizes (5). 

Evidently there are at least two compartments containing free labelled L-Phe taken up from the nutrient solution. One rapidly equilibrating with exogenous amino acid is directly linked with the incorporation of L-Phe into high molecular weight material (cytosolic pool). The second has a very high capacity, equilibrates only after 2-3 hours (results not shown in Pig. 2) and has no direct influence on rates of Phe-incorporation... 

Cytosolic compartments (peripheral pool internal pool) equilibrate rithin 4 min. with exogenous L-Phe. Tentative separation of two cytosolic pools accounts for the rapid pulse labelling of proteins even at very low concentrations of exogenous L-Phe in contrast to the lacking labelling under chase conditions. Both pools cannot be discriminated by the kinetic experiments (Pig. 2). 

Concommitant with equilibration of cytosolic pools protein synthesis proceeds linear after a lag period of 2 - 4 min. At very high levels of exogenous L-Phe (about 200 /Ug/ml) maximum 200 ng labelled L-Phe are incorporated into protein within 30 min. 3 his is the total amount of L-Phe needed for protein synthesis (value estimated from an average protein turnover of 8 %/hour). Evidently supply of L-Phe from intracellular sources must be shut down under these conditions. 

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